Steroid production with dactylium dendroides



tats

STEROID PRODUCTION WITH DACTYLIUM DENDROIDES William J. McAleer,Roselle, and Thomas H. Stoudt,

Westfield, N.J., assignors to Merck 8: Co., Inc., Rahway, N.J., acorporation of New Jersey No'Drawing. Original application Nov. 22,1957, Ser. No. 698,061. Divided and this application May 26, 1958, Ser.No. 737,549

4 Claims. (Cl. 195-51) Thisapplication is a division of our copendingapplication Serial No. 698,061, filed November 22, 1957, now

known compound, 16a-alkyl-4-pregnene3,20-dione having the. followingstructural formulaviii- .03

wherein R is alkyl.

In accordance with the present invention this starting material, namely,16u-alkyl-4-pregnene-3,20-dione, is subjected to the action of anoxidizing enzyme produced by an oxygenating strain of Dactyliumdendroides under aerobic conditions to formlfia-alkyl-l1u,17m-dihydroxy- 4-pregnene-3,20-dione, a compound havingthe following structural formulawherein R is as above.

The 16u-alky1-1let,17a-dihydroxy-4-pregnene-3 ,20-dione obtained abovemay be brominated at the 2l-position with bromine and chloroform to forml6u-alkyl-2l-bromo- 11a,l7a-dihydroxy-4-pregnene-3,20-dione. Uponreacting the latter compound with potassium acetate16aalkyl-l1m,l7a,2l-trihydroxy-4-pregnene-3,20 dione 21- acetate isformed. This latter compound is readily v--o on Patented Sept. 27,1960

ice

oxidized with sodium dichromate in acetic acid to 16:,-alkyl-17a,21-dihydroxy-4-pregnene-3,1 1,20-trione.

The l6a-alkyl-17u,21-dihydroxy-4-pregnene 3,11,20- trione can beconverted to te bis-semicarbazone, reduced with sodium borohydride andupon hydrolysis of the semicarbazone 16a-alkyl-l15,17a,21-trihydroxy-4-pregnene-3,20-dione is formed.

It has been found that bothl6a-alkyl-lla,l7a-dihydroxy-4-pregnene-3,20-dione andl6a-alkyl-1l,8,17a-21- trihydroxy-4-pregnene-3,ZO-dione possessextremely high anti-inflammatory activity.

In carrying out the process of the present invention, strains ofDactylium dendroides capable of affecting the oxygenation ofl6u-alkyl-4-pregnene-3,20-dione can be obtained from known culturecollections. For example, one such culture, Dactylium dendroides No.NRRL 2574 Y can be obtained from Northern Regional ResearchLaboratories, Peoria, Illinois. The microorganism is grown under aerobicconditions in a suitable nutrient medium in intimate contact with the16a-alkyl-4-pregnene-3,20- dione to be oxygenated; the fermentation orgrowing of the microorganism being continued until the desiredoxygenation has occurred. Thus, the 16ot-alkyl-4-pregnene- 3,20-dione tobe oxygenated can be incorporated directly in a suitable medium which isthen inoculated with an oxygenating strain of Daczylium dendroides andincubated under aerobic conditions thereby effecting the desiredoxygenation. Generally, our process is preferably effected by firstgrowing the microorganism in a suitable fermentation medium, then addingthe l6u-alkyl-4-pregnene-3,20-dione and continuing the cultivation ofthe microorganism under aerobic conditions for sufficient time to effectthe desired oxygenation.

The process of the present invention can be effected in both stationaryand submerged cultures of Dactylium dendroides growing under aerobicconditions, although, for practical purposes, it is most convenientlycarried out by growing the microorganism under submerged conditions in asuitable aqueous fermentation medium containing the steroid. The amountof the steroid which can be conveniently oxygenated by our method willdepend in part upon the particular medium employed. 5

Aqueous nutrient mediums suitable for the growing of oxygenating strainsof Dactylium dendroides must contain sources of assimilable carbon andnitrogen as Well as minor amounts of inorganic salts. Any of the usualsources of assimilable carbon such as dextrose, cerelose, glucose,inverted molasses, and the like, employed infermentation mediums can beused in carrying outthe process of our Invention. Similarly, complexsources of nitrogen usually employed in commercial fermentationprocesses such as lactalburnin digest (Edamin) and corn steep liquor, oran inorganic source of nitrogen such as sodium nitrate, ammoniumnitrate, and the like, are satisfactory for use in the fermentationmediums. Minor amounts of inorganic salts such as suitable soluble saltsof magnesium, potassium, sodium and iron are usually available incomplex sources of carbon and nitrogen or may be conveniently added tothe fermentation medium in minor amounts to promote maximum growth oftheoxygenating microorganism.

The following is an example of a suitable aqueous nutrient medium whichcan be used in our process of oxygenating steroids:

MEDIUM NO. 1

Dextrose 50.0

(NH HPO 7.5

K HPO 1.0

KCl 0.5

MgSO -7H O 0.5

FeSO -7H O 0.001

ZnSO -7H O Distilled water is added to give a total volume of 1 liter ofnutrient medium and the pH adjusted to 6.5 with sodium hydroxide.

The addition of minor amounts of anti-foaming agents, although notessential, is desirable with some fermentation mediums in order toobtain maximum yields of the desired oxygenated product. We have foundthat the addition to certain fermentation mediums of a substitutedoxazaline which is a nonvolatile, amine type, cationic surface activeagent available under the trade name Alkaterge C is particularlyeffective in reducing the amount of foam, although other anti-foamagents known to be useful for this purpose can also be used.

For example, 16a-methyl-4-pregnene-3,ZO-dione can be oxygenated inaccordance with the following procedure:

A sterile culture medium, such as those shown above is first inoculatedby introducing a small amount of spore suspension on vegetative growthof an oxygenating strain of Dactylium dendroides. The inoculatednutrient medium is then incubated at a temperature of about 27- 28" C.,while being agitated in the presence of oxygen for a period of about24-48 hours. At this point, a solution of16a-methyl-4-pregnene-3,20-dione in propylene glycol is added to thefermentation medium and the agitation and aeration of the nutrientmedium continued for about to 30 hours, or until the oxygenationreaction is completed.

When the oxygenation is complete, the oxygenated steroid may berecovered from the fermentation broth by extraction with a suitablewater immiscible organic solvent for the oxygenated steroids. Suitablesolvents for this purpose that might be mentioned are chloroform,methylene chloride, organic acid esters, aromatic hydrocarbons, and thelike. The solvent solution containing the desired oxygenated steroid canthen be evaporated to yield the desired product which may be furtherpurified by recrystallization or other procedures conventional in theart.

The 160L-alkyl-110t,17d dihydroxy 4 pregnene 3,20- dione obtained abovemay be brominated at the 21- position with bromine and chloroform toform 16a-alkyl- 21-bIOn10-1106,170t-dlhYdI'OXY 4 pregnene 3,20 dione.Upon reacting the latter compound with potassium acetate16m-alkyl-11u,17et,21-trihydroxy 4 pregnene 3,20- dione 2l-acetate isformed. This latter compound can then be oxidized with sodium dichromatein glacial acetic acid to form 16tx-alkyl-17a,21-dihydroxy-4-pregnene-3,11,20-trione.

The 16m-alkyl-l7a,21-dihydroxy 4 pregnene-3,l1,20- trione can beconverted to the bis-semicarbazone, reduced with sodium borohydride andupon hydrolysis of the semicarbazone 16a-alkyl-11B,17a,21-trihydroxy 4pregnene-3,20-dione is formed.

The following examples illustrate methods of carrying out the process ofthe present invention.

Example 1 Approximately 3.2 liters of nutrient medium No. 1 shown aboveand the minimum quantity of a substituted oxazaline (Alkaterge C)required to prevent foaming was sterilized for /2 hour at 100 C. Aftersterilization the medium was inoculated with approximately 250 ml. of avegetative growth of an oxidizing strain Dactylium dendroides cultureNRRL 2574 in the collection of the Northern Regional ResearchLaboratories, Peoria, I1-

4 linois. The mixture was then agitated using a 2 turbine type agitatorat 560 r.p.m. and air was passed in at a rate of 3 liters per minutemaintaining the temperature at 28 C. for a period of approximately 24hours.

At the end of the 24 hour period 1.28 g. of 16a-methyl-4-pregnene-3,20-dione dissolved in ml. propylene glycol was added to thefermented medium and agitation of the medium was continued at the same.Aeration of the medium was continued at 3.0l/minute for six hours andthen interrupted for six hours. This procedure was continued for aperiod of thirty-six hours following addition of the steroid.

Crystalline 1 1a,17ot-dihydroxy-16et-methyl-4-pregnene- 3,20-dione wasisolated from the fermented medium by extraction with ethyl acetateevaporation of the organic solvent in vacuo and recrystallization of theresidual material from ethyl acetate.

It should be understood that various changes may be made in the presentprocess as herein described without aflfecting the results attained.Thus, various modifications of conditions as to time, temperature,alkalinity, acidity, etc. and various changes in procedure differingfrom those herein given as illustrative of the preferred embodiments ofthis invention may be made without departing from the scope thereof.Accordingly, the scope of this invention is to be determined inaccordance with the prior art and the appended claim.

We claim:

1. The process for the production of 16a-alkyl-1la,17a-dihydroxy-4-pregnene-3,20 dione which comprises subjectingl6a-alkyl-4-pregnene-3,20-dione to the action of an oxidizing enzymeproduced by an oxygenating strain of Dactylium dendroides (NRRL 2574)under aerobic conditions.

2. The process for the production of 160t-I1'lCthy1-110t,17a-dihydroxy-4-pregnene-3,20-dione which comprises subjecting16ot-methyl-4-pregnene-3,20-dione to the action of an oxidizing enzymeproduced by an oxygenating strain of Dactylium dendroides (NRRL 2574)under aerobic conditions.

3. A process which comprises growing an oxygenating strain of Dactyliumdendroides (NRRL 2574) in an aqueous medium containing sources ofassimilable carbon and nitrogen under aerobic submerged conditions inintimate contact with 16oc-alkyl-4-pregnene-3,20-dione to produce16ot-alkyl-11a,17a-dihydroxy-4-pregnene-3,20- dione.

4. A process which comprises growing an oxygenating strain of Dactyliumdendroides (NRRL 2574) in an aqueous medium containing sources ofassimilable carbon and nitrogen under aerobic submerged conditions inintimate contact with 16u-methyl-4-pregnene-3,20dione to produce 16amethyl-11a,17a-dihydroxy-4-pregnene-3,20- dione.

References Cited in the file of this patent UNITED STATES PATENTS2,602,769 Murray July 18, 1952 2,762,747 Murray Sept. 11, 1956 2,844,513Wettstein et a1. July 22, 1958 OTHER REFERENCES Meister et al.:J.A.C.S., 76, Aug. 3, 1954, pages 4050 and 4051.

1. THE PROCESS FOR THE PRODUCTION OFF 16A-ALKYL-11A,17A-DIHYDROXY-4-PREGNENE-3,20-DIONE WHICH COMPRISES SUBJECTING16A-ALKYL-4-PREGNENE-3,20-DIONE TO THE ACTION OF AN OXIDIZING ENZYMEPRODUCED BY AN OXYGENATING STRAIN OF DACTYLIUM DENDROIDES (NRRL 2574)UNDER AEROBIC CONDITIONS.